摘要
为建立检测蓝舌病病毒(BTV)和流行性出血病病毒(EHDV)的快速诊断方法,笔者根据BTV的第10基因节段(Seg-10)和EHDV的第5基因节段(Seg-5)序列,分别设计BTV与EHDV的特异性引物和TaqMan探针,经过反应条件优化,建立了同时检测两种病毒的双重荧光定量RT-PCR方法,并进行了特异性、灵敏度、重复性和临床样品检测验证。用该方法对BTV Seg-10和EHDV Seg-5节段的体外转录ssRNA为模板绘制标准曲线,相关系数(R^2)均大于0.995,线性关系较好;该方法可检测不同血清型的BTV和EHDV,与阿卡斑病病毒(AKAV)、中山病病毒(CHUV)、小反刍兽疫病毒(PPRV)、口蹄疫病毒(FMDV)和牛流行热病毒(BEFV)无交叉反应,表明该方法特异性强;对2种标准品的检出极限均为10 copies/uL,比常规RT-PCR敏感性高10倍;组内和组间重复试验的变异系数均小于2%;用所建立的双重荧光定量RT-PCR方法及常规RT-PCR方法,对100份血液样品和50份病毒液分别进行检测,符合率均大于90%。以上结果表明所建立的双重荧光定量RT-PCR方法具有快速、准确、敏感和稳定等优点,为BTV和EHDV感染的早期快速诊断、高通量检测及流行病学调查提供了有效的技术方法。
The aim of this study was to develop a duplex Taq Man real-time RT-PCR(q RT-PCR)assay for simultaneous detection of bluetongue virus(BTV)and epizootic hemorrhagic disease virus(EHDV).Under the optimized reaction conditions,the Taq Man RT-PCR was established with the specific primers and probes designed according to the Seg-10 of BTV and Seg-5 of EHDV.In result,the established assay possessed a good linear relationship and the correlation coefficient of standard curve were greater than0.995.The assay was highly specific for amplification of BTV and EHDV,and no amplification from Akabane disease virus,Chuzan virus,Peste des petits ruminants virus,foot-and-mouth disease virus and bovine ephemeral fever virus.The detection limit of the real-time PCR was 10 copies/u L for both BTV and EHDV,which was 10 times higher than the conventional RT-PCR in sensitivity.The coefficient of variation of this assay was less than 2%in both intro-and intra-groups.A total of 100 bloods and 50 virus samples were tested by the established assay and the conventional RT-PCR,and the coincidental rate were both above 90%between the two assays.In conclusion,this assay was convenient,strong-specific and high-sensitive,which could be applied to a rapid detection and identification of BTV and EHDV.
作者
杨振兴
朱建波
李占鸿
李卓然
何于雯
谢佳芮
李华春
廖德芳
杨恒
YANG Zhen-xing;ZHU Jian-bo;LI Zhan-hong;LI Zhuo-ran;HE Yu-wen;XIE Jia-rui;LI Hua-chun;LIAO De-fang;YANG Heng(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Veterinary and Animal Science Institute,Kunming 650224,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2019年第9期1104-1111,共8页
Chinese Veterinary Science
基金
国家重点研发计划项目（2017YFC1200505,2016YFD0500908）
云南省中青年学术和技术带头人后备人才培养项目（2017HB055）
作者简介
第一作者:杨振兴(1986-),男,云南昆明人,助理研究员,硕士,主要从事牛羊虫媒病毒研究,E-mail:s300yn@163.com;第一作者:朱建波(1970-),男,云南红河人,研究员,硕士,主要从事动物病毒学研究,E-mail:zhujb70@126.com;通信作者:廖德芳,研究员,硕士,主要从事动物虫媒病毒研究,E-mail:wenjie3@hotmail.com;通信作者:杨恒,研究员,博士,主要从事动物病毒学研究,E-mail:yangheng2008.cool@163.com。
